Immunotherapy against murine leukemia

Three distinct digestive protease systems were induced in larvae of the herbivorous pest, Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), and used as a model to assess the ability of the proregion of papaya proteinase IV (PPIV; glycyl endopeptidase, EC 3.4.22.25) to act as an inhibitor of insect digestive cysteine proteinases. As shown by gelatin/PAGE and complementary inhibition assays, a recombinant form of the proregion produced in Escherichia coli inhibited a fraction of the insect proteases also inhibited by the well-characterized inhibitor of cysteine proteinases, oryzacystatin I (OCI). In contrast with OCI, the inhibitory potency of the proregion was affected by an increase of the temperature, suggesting a certain alteration of its structural integrity by the insect non-target proteases. This apparent susceptibility to proteolysis was confirmed by SDS-PAGE, after challenging the proregion with the different insect extracts. As seen on gel, selective inhibition of the insect aspartate proteinase, cathepsin D, with the inhibitor pepstatin A preserved the activity of the proregion against cysteine proteinases by preventing its hydrolysis. Taken together, these observations suggest the potential of plant protease proregions as regulators of cysteine proteinases in biotechnological systems, and show the ability of protease inhibitors to preserve the integrity of 'companion' defense-related proteins from the action of insensitive proteases in target pests.     

The in vitro uptake of albumin by adult Haemonchus contortus is altered by extracorporeal digestion

FITC (fluorescein isothiocyanate)-labeled albumin was used as a substrate to further characterize the digestion of host blood proteins by adult Haemonchus contortus. Isolated H. contortus intestine degraded FITC-albumin; degradation was completely inhibited by E-64, a specific inhibitor of cysteine proteases. The in vitro uptake and degradation of FITC-albumin by parasites was also evaluated. Uptake of fluorescence was demonstrated; greater than 50% of the fluorescence was associated with degraded FITC-albumin. However, both the uptake and degradation of FITC-albumin were reduced by about 85%. Additionally, when parasites were incubated in the presence of specific inhibitors of cysteine proteases, degradation of FITC-albumin was shown to occur rapidly in the media during in vitro incubation. This degradation was blocked completely by the presence of cysteine protease inhibitors. These results indicate that the rapid extracorporeal digestion of FITC-albumin (mediated by secreted cysteine proteases) produce relatively high concentrations of low molecular weight FITC-labeled fragments and alters the kinetics of uptake of fluorescence by the parasite. These fragments rather than FITC-albumin are rapidly taken up by the parasites. Thus in vitro demonstration of a definitive role for the cysteine proteases as intestinal digestive enzymes in H. contortus is compromised by the enzyme's extracorporeal activity. This extracorporeal cysteine protease activity may represent the enzyme's predominant activity.     

Molecular cloning, characterization and overexpression of two distinct cysteine protease cDNAs from Leishmania donovani chagasi

We have cloned and characterized two distinct cysteine protease cDNAs from Leishmania donovani chagasi. One of the cDNAs, Ldccy2, was isolated from a cDNA library prepared from total promastigote RNA while the other cDNA, Ldccys1, was isolated from a cDNA library prepared from total amastigote RNA. Ldccys2 has an open reading frame of 471 amino acids and Ldccys1 has an open reading frame of 447 amino acids. Comparison of the predicted protein sequences of the two distinct cysteine proteases with those of cysteine proteases from Leishmania pifanoi, a member of the L. mexicana complex, showed that the cysteine proteases from the two species of Leishmania are similar in their protein sequences. Each of the two cDNAs is distinct in genomic arrangement and chromosome location. Ldccys1 belongs to a family of cysteine proteases encoded by tandemly organized genes located on chromosome 7 while Ldccys2 appears to be a single cysteine protease gene located on chromosome 10. The organization of the two families of cysteine protease genes in L. donovani donovani was also found to be similar. In this species, the Lddcys1 genes are located on chromosome 5 while the Lddcys2 gene is located on chromosome 8. The Ldccys1 genes are expressed abundantly in the amastigotes recovered from infected hamsters, but at a very low level in the promastigote stage of development. On the other hand, the Ldccys2 gene is expressed both in the promastigote and amastigote stages. We have overexpressed the two cDNAs of cysteine proteases in Leishmania cells and the over-produced cysteine proteases are biologically active and are inhibited by cysteine protease inhibitors. Furthermore, the over-produced and indigenous amastigote specific cysteine protease, Ldccys1, reacted with polyclonal antibodies raised against this protein.     

Cytokine dysregulation in the post-Q-fever fatigue syndrome

High levels of protease inhibitors are induced in potato leaves by wounding. These inhibitors, when ingested by Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, induce expression of specific proteolytic activities in the gut. Induced protease activities cannot be inhibited by potato inhibitors and thus enable the insects to overcome this defence mechanism of potato plants. The induced aminopeptidase and endoproteolytic activities both have the characteristics of cysteine proteases. Twenty-one protein inhibitors of different structural types have been examined for their ability to inhibit these activities in vitro. Members of the cystatin superfamily were found to be poor inhibitors of the induced endoproteolytic activities, except for the third domain of human kininogen, which was a fairly strong inhibitor (75% inhibition). The strongest inhibition (85%) of induced endoproteolytic activity was obtained using structurally different thyroglobulin type-1 domain-like inhibitors--equistatin and MHC class II-associated p41 invariant fragment. Experiments performed using three synthetic substrates for endoproteases gave similar results and indicate the existence of at least different endoproteolytic enzymes resistant to potato inhibitors. The induced aminopeptidase activity can be inhibited only by stefin family of inhibitors in cystatin superfamily. In in vivo experiments, Colorado potato beetle larvae fed on equistatin-coated potato leaves were strongly retarded in their growth and almost 50% died after 4 days. This demonstrated the potential of equistatin to protect crops from insect attack.     

Bax cleavage is mediated by calpain during drug-induced apoptosis

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.     

Submandibular salivary proteases: lack of a role in anti-HIV activity

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.     

Protease IV, a unique extracellular protease and virulence factor from Pseudomonas aeruginosa

Comparisons of virulence between a Pseudomonas parent strain and an isogenic mutant devoid of protease IV have demonstrated a significant role for this enzyme during infection. We have characterized purified Pseudomonas aeruginosa protease IV in terms of its biochemical and enzymatic properties, and found it to be a unique extracellular protease. The N-terminal decapeptide sequence of protease IV is not homologous with any published protein sequence. Protease IV has a molecular mass of 26 kDa, an isoelectric point of 8.70, and optimum enzymatic activity at pH 10.0 and 45 degreesC. Purified protease IV demonstrates activity for the carboxyl side of lysine-containing peptides and can digest a number of biologically important proteins, including immunoglobulin, complement components, fibrinogen, and plasminogen. Protease IV is not inhibited by thiol-, carboxyl-, or metalloproteinase inhibitors. The total loss of enzyme activity in the presence of N-p-tosyl-L-chloromethyl ketone and the partial inhibition of enzyme activity by diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride imply that protease IV is a serine protease. Inhibition by dithiothreitol and beta-mercaptoethanol suggests that intramolecular disulfide bonds are essential for enzyme activity. The characteristics of this enzyme suggest that inhibitors of serine proteases could be developed into a medication designed to arrest tissue damage during Pseudomonas infection.     

Antifungal activity of interleukin-2-activated natural killer (NK1.1+) lymphocytes against Candida albicans

Protease inhibitors are currently the most effective antiviral agents against human immunodeficiency virus type 1 (HIV-1). In this study we determined the effect of four HIV-1 protease inhibitors on human T cell leukemia virus type 1 (HTLV-I). Rhesus monkey cells infected with HTLV-I were treated with different concentrations of indinavir, saquinavir, ritonavir, or nelfinavir. The effect of these inhibitors was monitored through their effect on the processing efficiency of the viral Gag protein in cells, the natural substrate for the viral protease. These inhibitors failed to block processing of HTLV-I Gag. To confirm these findings, human cells were cotransfected with plasmids encoding infectious copies of HIV-1 and HTLV-I, and the cells were subsequently treated with these same HIV-1 protease inhibitors. At concentrations between 5 and 50 times the IC50 for inhibition of HIV-1 replication, inhibition of HIV-1 Gag cleavage was apparent. In contrast, no effect on HTLV-I Gag processing was seen. At higher concentrations, HIV-1 Gag processing was essentially completely inhibited whereas HTLV-I Gag cleavage was still unaffected. Thus, these inhibitors are not effective inhibitors of HTLV-I Gag processing. Sequence alignments of the HIV-1 and HTLV-I viral proteases and processing sites suggest that the active site of the HTLV-I protease may have subtle differences in substrate recognition compared with the HIV-1 protease.     

Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro

The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to examine the biochemical changes that are involved when germ cells are cocultured with Sertoli cells in vitro preceding the establishment of specialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and basal compartments of bicameral units were collected to assess serine and cysteine protease activity. The expression of selected serine and cysteine proteases and their corresponding inhibitors in these Sertoli-germ cell cocultures was also examined by RT-PCR. Using an [125I]-collagen film assay, a transient but significant increase in serine protease activity was noted in both the apical and basal compartments when germ cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions. A specific tryptase (RNK-Tryp 2, a serine protease formerly cloned from a rat granular lymphocyte leukemia cell line, RNK-16, cDNA expression library) was shown to be expressed exclusively by Sertoli cells and not germ cells. Furthermore, Sertoli cell tryptase expression as well as urokinase plasminogen activator (u-PA, also a serine protease) increased significantly when germ cells were adhering to Sertoli cells. The decline in total serine protease activity when Sertoli-germ cell junctions were being formed was accompanied by a concomitant increase in alpha2-macroglobulin (alpha2-MG, a nonspecific protease inhibitor) expression. No significant changes in cysteine protease activity in either the apical or basal compartment were noted. However, there was a transient but significant increase in cathepsin L expression when germ cells were adhering to Sertoli cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results suggest that proteases and their corresponding inhibitors are working synergistically and are likely to be involved in the adherence of germ cells to Sertoli cells and the subsequent formation of intercellular junctions.     

Structure-based design, synthesis and evaluation of conformationally constrained cysteine protease inhibitors

The inhibition of cysteine proteases is being studied as a strategy to combat parasitic diseases such as Chagas' disease, leishmaniasis, and malaria. Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiologic agent of Chagas' disease. A crystal structure of cruzain, covalently inactivated by fluoromethyl ketone inhibitor 1 (Cbz-Phe-Ala-FMK), was used as a template to design potential inhibitors. Conformationally constrained gamma-lactams containing electrophilic aldehyde (12, 17, 18, 25, 26, and 29) or vinyl sulfone (43, 44, and 46) units were synthesized. Constrained lactam 26 had IC50 values of ca. 20 nM against the Leishmania major protease and ca. 50 nM versus falcipain, an important cysteine protease isolated from Plasmodium falciparum. However, all of the conformationally constrained inhibitors were weak inhibitors of cruzain, compared to unconstrained peptide aldehyde (e.g. 5 ) and vinyl sulfone inhibitors (e.g. 48, which proved to be an excellent inhibitor of cruzain with an apparent second order inhibition rate constant (k(inact)/Ki) of 634,000s(-1)M(-1). A significant reduction in activity was also observed with acyclic inhibitors 30 and 51 containing alpha-methyl phenylalanine residues at the P2 position. These data indicate that the pyrrolidinone ring, especially the quarternary center at P2, interferes with the normal substrate binding mode with cruzain, but not with falcipain or the leishmania protease.     

Peptidomimetic design

Major discoveries have been made of new type-I and type-III peptidomimetic inhibitors of peptide-derived systems. Innovative reversible inhibitors of cysteine proteases and renin, and additional examples of peptidomimetic inhibitors of interleukin-1 beta-converting enzyme, neutral endopeptidase, herpes simplex virus protease, thrombin, HIV protease, Ras farnesyltransferase, the RGD motif, Factor Xa and various aspartic proteases have been discovered.     

Structure-based inhibitor design by using protein models for the development of antiparasitic agents

The lack of an experimentally determined structure of a target protein frequently limits the application of structure-based drug design methods. In an effort to overcome this limitation, we have investigated the use of computer model-built structures for the identification of previously unknown inhibitors of enzymes from two major protease families, serine and cysteine proteases. We have successfully used our model-built structures to identify computationally and to confirm experimentally the activity of nonpeptidic inhibitors directed against important enzymes in the schistosome [2-(4-methoxybenzoyl)-1-naphthoic acid, Ki = 3 microM] and malaria (oxalic bis[(2-hydroxy-1-naphthylmethylene)hydrazide], IC50 = 6 microM) parasite life cycles.     

Evidence for participation of a calpain-like cysteine protease in cell cycle progression through late G1 phase

Recent studies have demonstrated that cell-permeant protease inhibitors arrest human fibroblasts in late G1. The target for the inhibitors has been claimed to be either the proteasome, or a calpain-like cysteine protease activity. In the present investigation, the progression of serum-stimulated WI-38 fibroblasts into S-phase was partially inhibited by the cell-permeant general inhibitor of cysteine proteases, E64d, but not by its non-permeant anolog, E64c. Exposure of fibroblasts in late G1 to the proteasome inhibitor, lactacystin, produced only a modest inhibition of progression into S-phase, and did not influence the extensive inhibition produced by the calpain-selective inhibitor, ZLLY-DMK. ZLLnV-CHO and ZLLL-CHO, which are reportedly selective for the proteasome, were less potent than ZLLY-DMK as inhibitors of S-phase progression. These results argue for the involvement of a calpain-like protease acting in late G1 to allow transit into S-phase.     

Protease activities during preparation and handling of nuclear particles containing hnRNA

Conditions were devised to avoid protease activity during the preparation and the subsequent handling of nuclear particles containing hnRNA. During all the steps of preparation of rat liver particles, the presence of phenylmethyl sulfonyl fluoride (PMSF) was required for the reproducibility of the results. It probably inhibited the cellular serine proteases before the separation of the particles from the other cellular structures. Protease activity was detected in the rat liver particles. The enzyme(s) preferentially hydrolyzed a few particle polypeptides. It was not inhibited by PMSF, nor by two trypsin and chymotrypsin-like protease inhibitors, nor by iodoacetamide, but was inhibited by sodium bisulfite and para-hydroxymercury benzoate (PHMB). PHMB was preferred above bisulfite because it could be used at lower concentration. It proved useful when particles were to be incubated at 37 degrees C. A protease hydrolysing the same polypeptides as the liver enzyme was also detected in rat brain particles. However, its activity was much lower in this tissue and the presence of protease inhibitors was not absolutely required under the standard conditions of preparation and handling of brain particles.     

The hypnic ("alarm clock") headache syndrome

Dipeptidyl peptidase IV (DPP IV, CD 26) is an integral membrane serine protease exhibiting a well characterized exopeptidase activity. The present study shows that DPP IV also possesses a novel gelatinase activity and therefore endopeptidase activity, which was directly demonstrated by gelatin zymography. Protease inhibitor profile analysis showed that the endo- and exopeptidase activities of DPP IV share a common active site. Substrate specificity was detected for denatured collagen types I, II, III and V suggesting that DPP IV might contribute to collagen trimming and metabolism. On the basis of these data we propose that DPP IV and the recently sequenced gelatinolytic seprase (FAPalpha) represent a new subfamily of gelatinolytic integral membrane serine proteases.     

Protease inhibitors for the treatment of human immunodeficiency virus infection

The pharmacology, pharmacokinetics, efficacy, adverse effects, drug interactions, and dosage and administration of protease inhibitors are reviewed. Protease inhibitors are a novel class of drugs used for the treatment of human immunodeficiency virus (HIV) infection. Saquinavir, ritonavir, indinavir, and nelfinavir have been approved in the United States; several other agents are under development. Protease inhibitors selectively block HIV protease, an enzyme involved in the later stages of HIV replication. Various pharmacokinetic differences exist among these agents, including differences in bioavailability, protein binding, and drug interactions. The drugs undergo extensive hepatic metabolism; dosage adjustments should be considered for patients with hepatic dysfunction. Clinical trials have shown protease inhibitors to be effective in reducing HIV RNA levels and increasing CD4+ lymphocyte counts. When protease inhibitors are used in combination with other antiretroviral agents, an additional beneficial effect on these markers occurs. Adverse effects of saquinavir and nelfinavir include mild gastrointestinal disturbances such as diarrhea. Ritonavir is less well tolerated because of gastrointestinal disturbances and circumoral and peripheral paresthesia. Indinavir has been associated with nephrolithiasis and asymptomatic hyperbilirubinemia. The development of resistance to protease inhibitors may be related to suboptimal dosages, noncompliance, or partial compliance. Protease inhibitors are potent and highly selective agents that block a critical step in HIV replication. They are effective and relatively well tolerated, but they are expensive, have extensive drug interaction profiles, and require careful compliance with the prescribed regimen.     

Inhibition of Entamoeba histolytica proteolytic activity by human salivary IgA antibodies

Entamoeba histolytica is a protozoan parasite that causes amoebiasis in humans; as the infection occurs mainly in the intestinal epithelium, the secretory immune response of the host could have an influence on the outcome. Secretory IgA antibodies against E. histolytica have been detected in asymptomatic and symptomatic patients, but little is known about their protective role. E. histolytica cysteine proteases seem to be involved in the pathogenesis of amoebiasis; therefore, it is important to evaluate the human IgA response against these proteases and its effect on their enzymatic activity. When human saliva samples with and without antibodies against E. histolytica were tested by Western blot against one purified 70 kDa amoebic cysteine protease, 84% of anti-amoeba-positive samples recognized it. The secretory IgA purified from a pool of anti-protease-positive samples had a strong in vitro inhibitory effect on the E. histolytica proteolytic activity. These results suggest that this effect, if it occurs in vivo, could be an important protective factor against this parasite.     

Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burden and egg production in vivo

Schistosome parasites utilize hemoglobin as a major protein source for their metabolism. Degradation of hemoglobin has been hypothesized to be mediated by both cysteine and aspartyl proteases secreted into the lumen of the parasite intestine. We now show that two distinct types of irreversible cysteine protease-specific inhibitors both arrest schistosome hemoglobin degradation in vitro. Arrest of hemoglobin degradation is followed by death of developing schistosomula 1 week later. Schistosome infected mice treated by a dose of 2 mg inhibitor per day for 1 week early in infection, and 2 weeks at the time of egg production, showed a significant reduction in worm burden, hepatomegaly, and the number of eggs produced per female worm. Histopathology showed a minimal immune response to those eggs which were produced, consistent with a delay in egg production relative to untreated infections. By tagging the inhibitor with biotin, specific cysteine protease targets were identified in extracts of schistosome worms.     

Human immunodeficiency virus type 1 protease inhibitors: evaluation of resistance engendered by amino acid substitutions in the enzyme's substrate binding site

The human immunodeficiency virus type 1 (HIV-1) protease is a homodimeric aspartyl endopeptidase that is required for virus replication. A number of specific, active-site inhibitors for this enzyme have been described. Many of the inhibitors exhibit significant differences in activity against the HIV-1 and HIV type 2 (HIV-2) enzymes. An initial study was conducted to ascertain the HIV-1 protease's potential to lose sensitivity to several test inhibitors while retaining full enzymatic activity. The substrate binding sites of the HIV-1 and HIV-2 enzymes are almost fully conserved, except for four amino acid residues at positions 32, 47, 76, and 82. Accordingly, recombinant mutant type 1 proteases were constructed that contained the cognate type 2 residue at each of these four positions. The substitution at position 32 resulted in a significant adverse effect on inhibitor potency. However, this substitution also mediated a noted increase in the Km of the substrate. Individual substitutions at the remaining three positions, as well as a combination of all four substitutions, had very little effect on enzyme activity or inhibitor susceptibility. Hence, the four studied active site residues are insufficient to be responsible for differences in inhibitor sensitivity between the HIV-1 and HIV-2 proteases and are unlikely to contribute to the generation of inhibitor-resistant mutant HIV-1 protease.